1. Field of the Invention
This invention relates generally to methods and compositions for direct detection of specific nucleic acid sequences associated with flanking regions of chromosomal aberration breakpoints, by forming hybrids between the sequences and genetic probes, and detecting the probes. In particular aspects, the invention concerns detection of nucleic acid sequences in situ in chromosomes, and more specifically in cells, including interphase cells. Compositions of probes useful for detecting chromosomal translocations, in particular those associated with human leukemias, are also disclosed.
2. Description of the Related Art
Substantial proportions of human diseases and malformations trace their etiology, at least in part, to genetic factors. Some of these factors are present in the zygote, others occur later as somatic cells form. Detection of genetic factors associated with particular diseases or malformations provides a means for diagnosis and treatment. For some conditions, early detection may allow prevention or amelioration of the devastating courses of diseases.
One class of genetic factors are chromosomal aberrations, that is, deviations in the expected numbers and structure of chromosomes for a particular species, and for particular cell types within a species. These may be constitutive i.e. present in the zygote, or induced post-zygotically in somatic (non-germinal cells) leading to mosaicism, that is a condition where both normal and abnormal cells are present. Chromosomes are the microscopically visible entities that are composed of the genetic material and, in higher organisms such as man, proteins and RNA. The study of chromosomes is called xe2x80x9ccytogeneticsxe2x80x9d.
There are several classes of structural aberrations that may involve autosomes or sex chromosomes or both. These aberrations are detected by noting changes in chromosome morphology (band patterns). The band patterns may be only changed in one chromosome (intrachromosomal) or in more than one chromosome (interchromosomal). Normal phenotypes may be associated with these rearrangements if the amount of genetic material has not been altered, but physical or mental anomalies are expected if there is gain or loss of genetic material. Simple deletions (deficiencies) refer to loss of part of a chromosome. Duplication refers to addition of material to chromosomes. Duplication and deficiency of genetic material can be produced by simple breakage of chromosomes, by errors during DNA synthesis, or as a consequence of segregation of other rearrangements into gametes.
Translocations are interchromosomal rearrangements effected by breakage and transfer of part of chromosomes to different locations. In reciprocal translocations, pieces of chromosomes are exchanged between two or more chromosomes. Generally, the exchanges of interest are between nonhomologues. If all the original genetic material appears to be preserved, this condition is referred to as balanced. Unbalanced forms have duplications or deficiencies of genetic material associated with the exchange; that is, something has been gained or lost xe2x80x9cin the shuffle.xe2x80x9d
One of the most exciting associations between chromosomal aberrations and human disease, is that between chromosomal aberrations and cancer. These aberrations are generally not constitutive, i.e., present in the zygote, therefore are not present in all cellsxe2x80x94only the abnormal ones. A mosaic condition is said to exist. For example, the Philadelphia (Ph1) chromosome is an important cytogenetic finding in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). This chromosome was originally identified as a chromosome sized slightly smaller than a xe2x80x9cG-groupxe2x80x9d chromosome. It was believed to be a deleted chromosome until detection of a reciprocal translocation between chromosomes No. 9 and 22 was reported by Rowley. A reciprocal translocation is one caused by breakage of at least two chromosomes and reunion of the broken piece in new locations.(FIG. 1) This aberration was first found to be associated with CML, but is now known to have prognostic and diagnostic value for many hematopoietic malignancies, e.g. ALL.
It is not only the translocation per se that is of clinical interest, but rather the resulting fusion of the proto-oncogene abl from the long arm of chromosome 9 with the bcr gene of chromosome 22, a consistent finding in CML. This genetic change leads to formation of a bcr-abl transcript that is translated to form a 210 kD protein present in virtually all cases of CML. This fusion can be detected by Southern analysis for bcr rearrangements or by in vitro amplification (PCR) of a complementary DNA (cDNA) transcript copied from CML mRNA. In approximately 95% of cases, the fusion gene results from a reciprocal translocation involving chromosomes 9 and 22, producing a cytogenetically distinct small acrocentric chromosome called Ph1. In the remaining cases the genetic rearrangement is more complex, and the involvement of the bcr and abl regions of chromosomes 9 and 22 may not be apparent during analysis of banded metaphase chromosomes. Southern blots, PCR, and metaphase chromosome banding analysis provide complementary, but incomplete, information on CML. They do not permit a genetic analysis on a cell by cell basis in a format in which the results can be related to cell phenotype as judged by morphology or other markers. Thus, assessment of the distribution of the CML genotype among cells of different lineage and maturity has not been possible.
As an example of the prognostic value of chromosomal aberrations, in adult ALL, the Ph1 chromosome is present in up to one-third of cases, and is associated with a high relapse rate and short survival. In pediatric ALL it is much less common, but it remains one of the few chromosomal abnormalities that continues to carry a poor prognosis in spite of newer, more intensive approaches to treatment. The accurate detection of the Ph1 is thus an important part of the diagnostic evaluation of patients with ALL.
Unfortunately, the cytogenetic diagnosis of the Ph1 chromosome in ALL has been limited. Cytogenetic analysis has a high failure rate in this disease, compared to other acute leukemias or to CML. Fewer than 70% of cases have adequately banded chromosomes at metaphases in most reports. xe2x80x9cBandingxe2x80x9d is a morphological pattern revealed by treating chromosomes to reveal horizontal stripes which vary in width and staining intensity and are characteristic of specific chromosomal regions. As an alternative to cytogenetic analysis, recently, newer methods of chromosomal in situ hybridization with non-isotopically labelled genetic probes have improved and extended the capabilities of cytogenetics. One of these methods is fluorescence in situ hybridization (FISH). In this method, probes are labelled with fluorescent signals that are detectable, generally by microscopic viewing of colors. Probes are nucleic acid sequences which bind to matching (homologous) sequences, e.g. on chromosomes. Although based on cytogenetic diagnosis, FISH may be performed on interphase cells as well as on metaphases, and may be applied directly to cells from either the peripheral blood or bone marrow without the need for banded karyotypes. The diagnostic utility of FISH with repetitive, centromeric probes in cases of leukemia has been demonstrated in previous studies.
FISH on interphase cells has proven to be a useful method for diagnosis and clinical management in hematologic diseases. However, much of this experience has concentrated on detecting numerical chromosomal abnormalities (single chromosome loss or gain), making use of chromosome-specific alpha satellite probes, which are highly-repetitive, unique sequences that occur within or near the centromere of chromosomes. The centromere is a constriction most readily visible at metaphase of cell division, which occurs at a characteristic location on each chromosome. The development of competitive hybridization methods to eliminate the signal from Alu-type repeats, and improvements in optics and reagents, have also made it possible to visualize single-copy genomic clones by FISH. However, the use of genomic clones is more difficult than the use of alpha satellite probes, because of lower signal intensity and high background. These difficulties would be offset if use of genomic clones produced improvements in disease assay specificity and were more flexible. Genomic clones are those that contain repeated sequences and non-coding sequences, that is DNA as it exists in the chromosome.
Some of the background for the present invention is as follows: single stranded synthetic DNA was developed with multiple sites are incorporated where fragments may be used as probes. (Stephensen, U.S. Pat. No. 4,681,840). Oncogenes are genes whose products have the ability to transform eukaryotic cells so that they grow in a manner analogous to tumor cells. Probes and methods for detecting chromosomal translocations are disclosed in EPO 181 635 (Groffen et al.)
Pinkel et al. (1986, 1988) and Gray et al. (1990) relate fluorescent-labeled probes for the cytogenetic analysis of chromosomes, and in situ hybridization of chromosomes at metaphase and interphase with whole chromosome-specific DNA.
In situ hybridization using a mixture of radioactive labelled probes c-abl and bcr sequences were employed on a CML patient sample. Although a translocation was said to be detected, Poisson analysis, a statistical procedure, was required to differentiate random from non-random silver grain distribution after autoradiography. (Bartram et al., 1987).
Benn et al. (1987) relates the molecular genetic analysis of the bcr rearrangement in the diagnosis of CML. Analysis involved Southern blots and radioactively labelled probes.
A single bcr-derived probe from which highly repetitive sequences were removed, was employed to detect the Ph1 translocation in CML. Restriction fragment length polymorphisms (RFLP) were used to identified patients affected with CML. Probes were used to map the chromosome 22 breakpoints within the bcr region by Grossman et al. (1989). Two separate bcr-specific probes were used to detect rearrangements within the bcr region. Southern blots and RFLP were employed. (Hutchins et al., 1989).
Flow cytometry has been applied to detection and characterization of diseasexe2x80x94linked chromosome aberrations (Gray et al., 1990). There is a great need to improve methods of detecting specific chromosome aberrations. Flow cytometry requires in vitro cell culture, expensive equipment, and expertise in interpretation of statistical analyses of results. Therefore, it is not generally clinically useful.
Detection of aberrations by use of repeat sequence probes found near centromeres, generally alpha satellite probes, or whole chromosome probes not probes specific for genetic regions associated with diseases. Greater sensitivity and increased resolution is needed. Use of whole chromosome probes is generally limited to detection of aberrations that occur homogeneously in a cell population (Gray et al., 1990) and does not have the resolution to distinguish similar, but distinct breakpoints. The present invention relates methods and compositions for detection of chromosomal aberrations that need not be present in all cells of a sample. Compositions include novel probes that were specifically designed to detect the BCR-ABL fusion gene in acute and chronic leukemias e.g. CML and ALL, and to determine molecular subtypes.
Methods using a plurality of probes to provide increased sensitivity and specificity in detecting chromosomal aberrations, are also aspects of the present invention. These methods are particularly valuable in being applicable to interphase cells, thus avoiding the costly, laborious, time-consuming and often inconclusive cytogenetic analysis of metaphase chromosomes, and the expertise needed for flow cytometry. Not only are the methods of the present invention easier to use, but these methods do not require invasive or risky techniques inflicted on patients, such as bone marrow sampling. However, the methods and compositions of the present invention may also be used on metaphase chromosomes or Souther blots.
Substantial proportions of human diseases and malformations trace their etiology, at least in part, to genetic factors. Some are inherited, some occur during the development and life of the organism. Cancers, for example, are associated with somatic mutations and/or chromosomal aberrations that may be specific for cancerous cells. Detection of genetic factors associated with particular diseases or malformations provides a means for diagnosis and treatment. For some conditions, early detection may allow prevention or amelioration of the devastating courses of diseases. For others, monitoring the course of the disease is useful to determine treatment strategies. The methods and compositions of the present invention provide multipronged reconnaissance into the genetic material to determine if it harbors abnormal factors.
This invention concerns genetic factors in the form of chromosomal aberrations, that is, deviations from the number and structure of chromosomes characterizing a species, and cell types within the species. In humans, for example, there are generally 46 chromosomes in somatic, i.e. non-germinal cells. These exist in 23 pairs, 22 of which are each matched by size and structural morphology. Structural morphology is revealed by a variety of methods, for example, treatment of chromosomes to form distinguishable horizontal bands. Analysis of such patterns, and comparison of the relative size of the chromosomes and positions of the centromere, a constriction visible at metaphase of the mitotic cell cycle on each chromosome, allow identification and classification of each pair. Analysis of banding patterns also permits detection of structural aberrations both between and within chromosomes.
For purposes of the present invention, structural chromosomal aberrations which comprise a breakpoint fusion region with nucleic acid sequences flanking the breakpoint fusion, are of particular interest. Flanking regions should be within 800 kb or less so that there are include a particular breakpoint, yet are far enough on either side of the fusion so that they are not included in it. An aspect of the present invention is to detection aberrations which are not detectable by conventional metaphase cytogenetics using light microscopy.
Chromosomes contain linear sequences of DNA, a nucleic acid that is the genetic determinative for most species (RNA is the genetic material in some lower organisms). Cloning technology has been developed which is capable of isolating specific genes directly from the genome.
To identify specific genes, that is, specific nucleic acid sequences, specific probes may be used that react only with the particular sequence of interest to seek it out from the vast excess of other sequences. The reaction of probes and their matching (homologous) sequences, is termed hybridizationxe2x80x94the joining of the probe and its match by hydrogen bonds. Laboratory methods related to cloning technology and other techniques well known to those of skill in the art, may be found in Maniatis (1982) and in Lewin (1987). Conditions of varying stringency are used depending on the degree of homology required for a match. In examples disclosed herein, stringency conditions are set forth that are specific for hybridization to unique breakpoint provided in preferred embodiments.
The power of this approach in cytogenetic analysis comes from the increasing availability of chromosome- or locus-specific-nucleic acid probes. These fall into three general classes: 1) probes for sequences that are present in many copies on one chromosome, 2) composite probes composed of many individual elements that are homologous to target sequences distributed more-or-less continuously along an entire chromosome, and 3) probes homologous to a specific chromosome subregion or locus; for example, associated with a genetic disease. To use probes to detect chromosomal aberrations formed by breakage and reunion of, e.g., two chromosomes from different pairs (non-homologous), by probing the sequence at the fusion of the breakpoints themselves, may provide a weak signal by which the hybridization is detected. This is because when a short sequence hybridizes, the signal may be too weak to be detected. If a probe is lengthened to provide greater signal intensity, it may become too large. This will be seen as a diffuse signal. Those of skill in the art will readily determine optimum probe size for a particular application using the guidelines disclosed herein.
A laboratory procedure that produces increased specificity and low background, and one in which individual probes can be distinguished as separate entities, is preferred. Thus, the labelling of probes is not amenable to current radioactive isotopic labels, but is more suitably performed with fluorescent and other non-isotopic, i.e. enzymatic or chemical labeling methods. For diagnosis using interphase cells, that stage of cell division that most somatic cells sampled clinically are in, these labelling methods provide good enough intensity to be detectable.
An upper limit on probe size for purposes of the present invention is believed to be about 200 kb of nucleic acids, that is, about 3 times the size used in the examples disclosed herein. A goal in determining suitable sizes for probes is to detect doublets. Doublets are pairs of distinct probes in closer proximity than expected based on there normal chromosome locations in the absence of aberrations. To overcome limitations inherent in some other techniques, this invention provides a strategy of multiple sorties into the genetic material using at least two probes for separate, but related sequences; for example, one for each of the flanking regions of a breakpoint at which fusion of two chromosomal segments has occurred. Moreover, this invention takes advantage of probes large enough to give an intense signal yet specifically targeted to a genomic sequence. To be distinguishable yet juxtaposed at interphase, labelled flanking regions have to be approximately within 800 kb.
The probes are preferably labelled so that their location in the genetic material may be determined. The location is generally determined by use of a microscope. To avoid increased time and the usual problems and risks associated with radioactive labels, fluorescent labels are preferred. A separate color for each independent probe provides the most information. e.g. red on one probe, green on the other.
The probes may be detected in situ, that is, without extraction of the genetic material. In general, it will be the cell, or, more specifically, the cell nucleus that will be viewed.
Although the cells may be analyzed in metaphase, a stage in cell division wherein the chromosomes are individually distinguishable due to contraction, the methods and compositions of the present invention are particularly useful for interphase, a stage in cell division wherein chromosomes are so elongated that they are entwined as is a bowl of spaghetti, and cannot be individually distinguished. At this stage the chromosomes may be referred to as chromatin.
An additional aspect of the invention is the use of genomic DNA fragments as probes, rather than fragments which correspond simply to transcribed/translated regions. By employing genomic fragments of up to 100 kb, e.g., through the use of cosmid clones, it is possible to obtain much greater relative degree of hybridization with the chromosomal DNA, a particular advantage where a light-microscopic detection is envisioned such as in the preferred method in the present invention.
Using multiple probes, each with a distinguishable label, the overall pattern of the probes is used to assay for a breakpoint. Because allelic genes exist on chromosome pairs, each labelled probe capable of hybridizing to a sequence normally present on a specific chromosome, appear twice. If one member of each of two chromosomes is involved in a translocation which moves the sequences hybridizing to the probes together on one fusion chromosome, two of the different colored probes will be in closer proximity to each other than expected if they maintain their original chromosome location, the other two will be more distant.
In particular aspects of this invention, specific disease entities are analyzed. The hematological malignancies provide illustrative embodiments as disclosed in the following sections. One of the most clinically useful assays for chromosomal aberrations is cytogenetic analysis directed at detection of the Philadelphia chromosome (Ph1) which is associated with chronic myelogenous leukemia (CML), and other hematological malignancies. The presence or absence of the Ph1 chromosome is a major diagnostic and prognostic aid. However, detection by cytogenetic analysis and other available techniques is time consuming, laborious, and not completely accurate.
An aspect of the present invention concerns the use of DNA probes for the direct detection of Philadelphia chromosomes in metaphase and interphase cells using non-radioactive methods. The so-called Philadelphia chromosome is a chromosomal aberration which results from a translocation between chromosome 9 and 22 which produces a longer chromosome 9 and shorter chromosome 22. The shortened chromosome 22, termed Ph1, is generally diagnostic of certain types of leukemia, including in particular chronic myelogenous leukemia (CML), as well as various other leukemias. On a molecular level, it has been shown that the development of the Ph1 chromosome includes a translocation of a portion of the c-abl oncogene into a breakpoint cluster region (bcr) of chromosome 22, which can activate the ABL gene.
A variety of molecular methods are known for diagnosing this abnormality in DNA or RNA extracted from cancer cells. The method of the present invention involves the use of a specific set of DNA probes, some corresponding to the abl gene, and some corresponding to the bcr gene. This specific set of probes is hybridized in situ to fixed cells of a sample from an individual suspected of being affected. The ABL and BCR specific probes are preferably labeled with separate fluorescein tags (e.g., biotin plus fluorescein-labeled avidin, or digoxigenin-labeled probes). Therefore, upon hybridization, both sets of labeled probes will hybridize to an a translocated chromosome 9 producing a two color doublet, whereas only the ABL specific probe will hybridize to chromosome 9 in non-affected individuals. The use of a visually detectable label allows a means of assessing the presence of the Ph1 chromosome through the application of light microscopy, providing a significant advantage in terms of expertise required to carry out the assay. The methods are simpler and more rapid than previously available.
Probes developed as an aspect of the present invention include three probes that are particularly useful for detection of hematopoietic malignancies, notably chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). The novel probes are designated: PEM12, c-H-abl and MSB-1. These probes are specific for regions of the BCR gene (MSB-1 and PEM12) and a region at the ABL gene (c-H-abl). The BCR and ABL regions are those which flank the breakpoint fusion region in the Ph1 chromosome associated with leukemias.
FISH was used to detect the Ph1 chromosome or its genetic equivalent as the fusion of BCR and ABL probes labeled with two colors. The method was successfully used in interphase cells of ALL patients. This method, using only two probes, only detects the p210 subtype of BCR-ABL gene fusions, whereas the majority of Ph1-chromosome-positive ALL cases contain the p190 fusion. For this reason, a combination of three probes used in pairs was developed that could detect both the p190 and p210 molecular subtypes. Methods using these combined probes are useful for Ph1 chromosome detection by FISH in ALL. Capabilities and limitations of probes and combinations of probes in the clinical setting were assessed and shown to provide improvements over previous assays for leukemias, in particular cytogenetic analysis of metaphase chromosomes. Although the embodiments herein relate to detection of chromosomal aberrations in leukemias, the probes may be specifically tailored to meet clinical needs for the diagnosis of any chromosomal aberration, as they have for translocations in leukemias. It is only necessary to be able to determine breakpoint regions and to develop probes to those regions.
The Philadelphia (Ph1) chromosome is also an important prognostic indicator in acute lymphocytic leukemia (ALL). Present in 30% of adult and 5% of pediatric cases, its presence portends a short remission duration and poor survival, despite improvements in therapy as in CML. It is a derivative of a translocation between chromosomes 9 and 22, and results in the fusion of a part of the ABL proto-oncogene on 9q with part of the BCR gene on chromosome 22. Molecular analysis shows it is much more heterogeneous because the BCR breakpoints are variable. Cytogenetic diagnosis of the Ph1 chromosome in ALL is possible in only 70% of cases because of the failure to obtain adequate metaphases. The new technique of fluorescence in situ hybridization (FISH) offers the advantage of allowing the diagnoses of chromosomal abnormalities in interphase cells, thus overcoming the problem of metaphase preparations.
Using dual-color FISH with probe combinations specifically tailored to flank the breakpoints in the two types of the BCR-ABL fusion genes p210 and p190, the presence or absence of the Ph1 chromosome in interphase cells was determined from 5 ALL patients, two ALL-derived cell lines, and normal lymphocytes and specified its molecular subtype when present. The method proved accurate for detection in all cases and for subtyping in 7 of 8 of the cases examined. The sensitivity and specificity for assessing the Ph1 status of individual cells were low, but results were unequivocal when several cells were examined in a sample.
As can be seen from the following descriptions and examples, the methods disclosed may be performed by a pathologist on routine examination of blood and tissue samples.